Effects of cycloheximide (CHX) on ribosome profiling experiments in Schizosaccharomyces pombe
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ABSTRACT: CHX is an inhibitor of translation elongation often used in ribosome profiling experiments. There is evidence that CHX treatment of cells may cause artefacts in the distribution of ribosomes on mRNAs. We investigate this possibility in S. pombe by performing ribosome profiling in the presence and absence of this drug.
Project description:We applied in parallel RNA-Seq and Ribosome-profiling to S. pombe pat1 diploids undergoing meiosis and sporulation in a synchronous manner
Project description:We applied in parallel RNA-Seq and Ribosome-profiling to S. pombe pat1 diploids undergoing meiosis and sporulation in a synchronous manner
Project description:Translation complexes are stabilised in vivo using formaldehyde. Cells are lysed, and cell extracts are treated with RNase I to produce protected RNA fragments (FPs or footprints). Extracts are then run through sucrose gradients to separate small ribosomal subunits and full ribosomes. FPs are isolated from each of these fractions and analysed by sequencing. Note this is a modified version of ribo-seq (a.k.a. ribosome profiling)
Project description:The Fil1 transcription factor regulates the response to amino acid starvation. Here we analyse the effects of overexpressing the fil1 coding sequence and of its deregulated expression. The following strains were used: 1] Strains expressing the fil1 gene under the control of the thiamine-repressible nmt1 promoter (nmt-fil1) 2] Strains in which 6 uORFs (upstream Open Reading Frames) in the fil1 5'-UTR have been inactivated, leading to deregulated translation of the fil1 mRNA (6uORF_mutant)
Project description:RNA-sequencing of S. pombe wild type, ppa1 mutants, and tor1 mutants, grown under condition of normal nitrogen and under nitrogen starvation.
Project description:To estimate mRNA steady-state levels we used RNA extracted from logarithmically growing fisson yeast cells on Affymetrix Yeast 2.0 Genechip arrays. The signal intensities from two independent biological repeats were averaged, resulting in measurements for 4818 out of 4962 nuclear protein-coding genes.